Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of the nerve growth factor (NGF) family of neurotrophins. The neurotrophins are polypeptides essential for the developing and adult brain; defects in their synthesis or ability to activate target cells may be important in several neurological diseases and in the aging normal brain. The receptors for this group of factors constitute the Trk family of receptor tyrosine kinases. BDNF activates specifically TrkB while NT- 3 binds preferentially to TrkC. The mechanism of action of these factors in the CNS is unknown and the purpose of this proposal is to investigate early signal transduction events initiated by BDNF and NT-3 in responsive neurons and astrocytes. Previous biochemical and molecular studies of neurotrophin action have relied heavily on transformed cell lines, that may not be an appropriate model for events that occur in post-mitotic central neurons. In situ hybridization studies have revealed that the mRNAs for BDNF and NT-3 are highly expressed in the developing and adult hippocampus. We have shown that cultured embryonic hippocampal neurons from rat brain express and respond to BDNF and NT-3. As these cultures consist almost purely of pyramidal neurons grown in physical isolation from co-cultured astrocytes, it is easy to separate the neuronal plane from the glial plane and perform biochemical experiments. The neurons have been shown to recapitulate an analogous developmental program in vitro as is observed in the intact animal. This system thus offers an excellent opportunity to study the mechanism of action of these neurotrophins in a pure population of primary neurons. We plan to investigate the tyrosine phosphorylation of substrate proteins using sensitive immunological techniques and to study downstream signal processing, particularly those events that involve protein kinases. Specifically, we will investigate the mechanism of raf and "MAP" kinase activation in these cells b y the two neurotrophins. These multifunctional enzymes appear to be important in mediating between external signals and transcriptional events in many cells. In parallel studies we will also examine the levels of expression of the neurotrophins themselves in developing hippocampal neurons. As these cells express both BDNF and NT-3, there may be an "autocrine" neurotrophic loop in this population of neurons. In other experiments we will examine the ability of BDNF and NT-3 alone, or in combination with other factors, to promote the development and survival of hippocampal neurons deprived of glial support, conditions under which they normally deteriorate and die. Finally, we have observed that CNS astrocytes in culture express a different form of the BDNF receptor, TrkB, than their neuronal counterparts. We suspect that this receptor is a truncated, non-catalytic form of TrkB that lacks the tyrosine kinase domain. However, these cells still signal in response to BDNF (MAP kinase is activated and c-fos is induced). We will investigate the signalling pathways used by this receptor and determine how they differ from those used by the full-length BDNF receptor found in neurons. These studies will contribute to our knowledge of neurotrophin action and shed light on how these molecules maintain neuronal viability in the intact brain.